Method of producing non-human mammals

ABSTRACT

A method of producing mutant/targeted non-human mammals, such as mutant mice that does not require production of chimera and permits the introduction of multiple mutations in embryos and, thus, avoids the necessity of breeding to combine all of the desired mutations in a single animal. The method is efficient in producing ES mice.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/755,003, filed Jan. 5, 2001, which claims the benefit of thefiling date of U.S. Provisional Application No. 60/234,378, filed Sep.20, 2000 and the filing date of U.S. Provisional Application No.60/255,970, filed Dec. 15, 2000. The entire teachings of the referencedapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] Work described herein was supported, in whole or in part, byNational Institutes of Health Grants No. 5-R35-CA44339 and RO1-CA84198.The United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] In the past two decades, considerable effort has been invested inproducing non-human mammals, such as mutant or transgenic mammals, suchas mice, and during that time, a variety of methods have been developed.In order to produce a desired mutant animal, such as a mouse, using anyof the presently available methods, one must first produce chimeras andbreed the chimeras to produce homozygous offspring; production ofoffspring which are not chimeric requires two breeding cycles for eachgene. This is the case for each mutation to be introduced and, ifoffspring exhibiting more than one mutation are desired, additionalbreeding cycles are required. For example, if mutant mice bearing sixdifferent alterations (e.g., six different genes) are to be produced,approximate breeding time will be two years. Producing desiredgenetically manipulated mammals, even those for which the breeding cycleis relatively brief, requires considerable time, as well as resources,using current methods. It would be very valuable if a more efficientmethod of producing mutant mammals, such as mice, were available.

SUMMARY OF THE INVENTION

[0004] The present invention relates to methods of producing non-humanmammals, which can be mutant non-human mammals or non-mutant non-humanmammals, such as mice, that do not require production of chimera orchimeric offspring (offspring that consist of cells that are derivedfrom more than one zygote). The invention is a method of producingnon-human mammals, such as mutant or non-mutant mice, by tetraploidblastocyst complementation using non-inbred pluripotent cells or celllines, such as non-inbred ES cells or cell lines.

[0005] The present method makes it possible to include multiplemutations or alterations in the same pluripotent cells (e.g., embryonicstem (ES) cells) or cell lines (e.g., ES cell lines) before producing ananimal from the ES cells or ES cell lines. The present invention relatesto methods of producing non-human mammals that, thus, avoid thetime-consuming step of breeding chimera to produce the desiredoffspring. As is evident from the work described herein, mutant ortargeted offspring, particularly mice, that are entirely derived from EScells or ES cell lines and survive postnatally have been producedwithout the need to produce chimeric intermediates. Mutations introducedinto the non-inbred pluripotent cells or cell lines can be non-random ortargeted alterations or can be random or non-targeted alterations. Theproducts of either approach are referred to herein as mutant. In thoseembodiments in which mutations are non-random or targeted, the resultingproducts can also be referred to as targeted (e.g., targeted ES cells,targeted ES cell lines, targeted non-human mutant mammals, such astargeted mutant mice). Alterations can be of a variety of types,including deletion, addition, substitution, or modification of all or aportion of DNA (e.g., a gene, regulatory element) in the ES cells. Thesealterations include addition of a gene or gene portion not normallypresent in the ES cells or ES cell lines. Non-mutant mice that arederived entirely from ES cells or ES cell lines and survive postnatallycan also be produced using the method described. The present methods ofproducing mice, particularly mutant mice, make it possible to produceoffspring, particularly mutant offspring, very efficiently, particularlyin comparison with other methods.

[0006] The present invention also relates to a method for derivingfertile XO female mice from non-inbred (F1) mouse male ES cells ornon-inbred (F1) mouse male cell lines and a method of deriving males andfemales carrying all genetic alterations introduced into a singlenon-inbred ES clone, such as a targeted non-inbred mouse ES cell clone.Breeding of the mutant males and females allows the production of amutant mouse strain derived from a single non-inbred ES cell clone, suchas a targeted (e.g., multiply targeted ES cell clone), withoutoutcrossing the mutant animal with a wildtype partner, as is required inpresently available methods.

[0007] The present invention also relates to non-human mammals,particularly mutant non-human mammals, such as mutant mice, produced bythe methods; cells obtained from the mutant or non-mutant non-humanmammals and cell lines produced from these cells. A particularembodiment is cells obtained from mutant or non-mutant mice produced bya method of the present invention; cells obtained from the mice and celllines produced from such cells. The invention further relates to amethod of producing blastocysts useful in the method of producing mutantor non-mutant mammals, such as mouse blastocysts (non-mutant or mutant)useful for producing non-mutant or mutant mice by the method describedherein and blastocysts produced by the method.

[0008] The invention also relates to methods of identifying XO F1 EScells (e.g., mouse XO F1 ES cells) comprising screening a population ofF1 ES cells, such as a population of wildtype or mutant F1 ES cells, forF1 ES cells that are XO F1 ES cells. In a particular embodiment,screening is carried out to identify F1 ES cells in which spontaneousloss of the Y chromosome has occurred, resulting in XO F1 ES cells. Bypopulation of F1 ES cells is meant a mixture of XO F1 ES cells and XY F1ES cells.

[0009] In particular embodiments, mutant non-human mammals (e.g., mutantmice) are produced to mimic or serve as a model for a condition (e.g., aneurological, muscular or respiratory condition, cancer, viralinfection, arthritis,) that occurs in another species, such as inhumans. They are used to identify new drugs that have a therapeutic orpreventive effect on the condition or assess the ability of known drugsto act as therapeutics or preventatives. Thus, the present inventionencompasses methods in which the mutant non-human mammals (particularlymutant mice) are used, such as in a method of screening to identify anew drug that inhibits the occurrence of (prevents the onset, reducesthe extent or severity of) or reverses a condition caused by orassociated with the genetic alteration(s) and a method of screeningknown drugs for those that inhibit onset of or reverse such conditions.Drugs identified by methods in which the mutant mammals of the presentinvention are used are also the subject of this invention. These includedrugs that inhibit onset of a condition (prevent the onset or reduce theextent to which the condition is established or severity of thecondition), referred to as preventatives or prophylactic drugs and drugsthat reverse (partially or completely) or reduce the extent or durationof the condition once it has occurred.

DETAILED DESCRIPTION OF THE INVENTION

[0010] As described herein, Applicants have demonstrated that geneticbackground is a crucial parameter controlling postnatal survival ofoffspring that are entirely derived from ES cells or ES cell lines. Thatis, heterozygosity of the genome of the pluripotent donor cell (e.g.,heterozygosity of the donor ES cell genome) is critical for postnatalsurvival of offspring whose development is achieved without thecontribution of normal cells derived from the host embryo. Further,Applicants have demonstrated that non-human mammals, particularly mice,can be generated without the need to first create a chimericintermediate. The ability to derive offspring (e.g., mice) directly fromES cells or ES cell lines without the need to produce chimericintermediates is a distinct advantage, not only because it avoids thetime consuming and expensive step of producing chimera, but also becauseit facilitates the generation of offspring with multiple geneticalterations. The generation of Fl ES cell-tetraploid mice provides asimple procedure for directly deriving animals with complex geneticalterations without the need to create a chimeric intermediate. Thetetraploid technology in combination with the use of F1 cells or F1 celllines allows assembly or production of multiple genetic alterations inthe same ES cell clone by consecutive gene targeting cycles in vitro.The resulting multiply targeted F1 ES cell clone is introduced intotetraploid blastocysts to produce an embryo that is then transferred toan appropriate foster mother and permitted to develop to term. Thus, atransgenic animal with one or multiple desired or selected geneticalterations can be generated without the need for production of chimericfounders and outbreeding with wild type mice.

[0011] Also described herein is a strategy for deriving fertile XOfemales from F1 male ES cells or F1 male ES cell lines and a method ofbreeding a mutant mouse strain derived from a given multiply targeted EScell clone without outcrossing the mutant animal with a wild typepartner. This avoids time consuming and costly outcrossing, which wouldotherwise be necessary. Because each F1 ES cell line is of a givensex—usually male—it would not be possible to breed a mutant mouse strainderived from a given multiply targeted ES cell clone withoutoutcrossing, using presently available methods. As described herein,however, outcrossing is no longer required, in view of the fact that itis possible to generate mutant males and females from a single targetedmale ES cell clone by selection for loss of one Y chromosome, resultingin generation of XO ES cells. In one embodiment, a negative selectionmarker (e.g., a negative selection gene, such as a Herpes Tk gene) isintroduced into the Y chromosome of F1 male ES cells, as describedfurther below, and the resulting cells are subject to selection with anagent (e.g., gancyclovir) which kills all cells carrying the Ychromosome. Cells that are not killed have lost the Y chromosome and,thus, are XO. This enables subsequent generation of males and femalescarrying identical genetic alterations, as described further below.

[0012] The invention described herein relates to a method of producingnon-human mammals, which can be mutant or non-mutant animals, such asmutant or non-mutant mice. As described herein, it has now been shownthat mutant non-human mammals can be produced without the intermediatestep of producing chimeric animals which, in presently availablemethods, must be bred to produce the desired mutants. In particular,targeted or mutant mice have been produced and the present invention isdescribed in detail by describing their production. However, the presentinvention is useful to produce mutants or non-mutants of any non-humanmammal for which embryonic stem (ES) cells can be obtained.

[0013] The invention is, in one embodiment, a method of producing anon-human mammal. The method comprises introducing non-inbredpluripotent cells, such as non-inbred ES cells, into tetraploidblastocysts of the same mammalian species, under conditions that resultin production of an embryo (at least one/one or more embryo) andtransferring the resulting embryo(s) into an appropriate foster mother,such as a pseudopregnant female of the same mammalian species. Theresulting female is maintained under conditions that result indevelopment of live offspring, thereby producing a mutant non-humanmammal. The resulting non-human mammal is derived from a single zygote(that which originally gave rise to the ES cells). Such mammals arereferred to herein as ES non-human mammals.

[0014] In another embodiment, the invention is a method of producing amutant non-human mammal. The method comprises introducing non-inbredpluripotent cells, such as non-inbred ES cells, comprising at least onemutation or alteration into tetraploid blastocysts of the same mammalianspecies, under conditions that result in production of an embryo (atleast one/one or more embryo) and transferring the resulting embryo(s)into an appropriate foster mother, such as a pseudopregnant female ofthe same mammalian species. The resulting female is maintained underconditions that result in development of live offspring, therebyproducing a mutant non-human mammal. The resulting mutant non-humanmammal is derived from a single zygote (that which originally gave riseto the ES cells). Such mammals are referred to herein as mutant ESnon-human mammals. The mutations or alterations can be non-random ortargeted or, alternatively, can be introduced randomly or in anon-targeted manner.

[0015] A specific embodiment of the present invention is a method ofproducing a targeted or mutant mouse, comprising: (a) introducing mousenon-inbred pluripotent cells comprising at least one alteration ingenomic DNA into mouse blastocysts, preferably tetraploid blastocysts,thereby producing mouse blastocysts containing mouse non-inbredpluripotent cells; (b) maintaining the product of (a) under conditionsthat result in production of embryos; (c) introducing an embryo orembryos (at least one/one or more embryos) into a foster mother, such asa pseudopregnant female mouse; and (d) maintaining the female into whichthe embryo(s) were introduced under conditions that result indevelopment of live offspring, thereby producing a mutant mouse. Themutant mouse is also referred to herein as a mutant ES mouse. In oneembodiment, the non-inbred mouse pluripotent cells are non-inbred mouseES cells, such as F1 cells derived from two different strains of mice orF2, F3 F4, etc. cells that can be derived from parents after consecutivebrother-sister matings. Alternatively, such cells can be derived fromparents after backcrossing an F1 strain to one of the parent strain toobtain the first backcross generation (N1) and by further backcrossingto obtain N2, N3, N4, etc. backcross generations. As used herein, theterm non-inbred ES cells encompasses all of the hereinabove described EScells and cell lines. Derivation of non-inbred ES cells, with specificreference to production of mouse ES cells, is described in detail in theExamples.

[0016] In a further embodiment, the invention is a method of producing anon-mutant mouse, referred to as a non-mutant ES mouse. The method iscarried out as described above for the production of mutant ES mice,except that DNA in the non-inbred pluripotent cells, such as non-inbredES cells (e.g., non-inbred mouse cells) has not been altered prior totheir use. That is, the non-inbred ES cells as obtained may containalterations or mutations, but are not further modified to producenon-random or random mutations. The method comprises: (a) introducingmouse non-inbred pluripotent cells into mouse blastocysts, preferablytetraploid blastocysts, thereby producing mouse blastocysts containingmouse non-inbred pluripotent cells; (b) maintaining the product of (a)under conditions that result in production of embryos; (c) introducingan embryo or embryos (at least one/one or more embryos) into a fostermother, such as a pseudopregnant female mouse; and (d) maintaining thefemale into which the embryo(s) were introduced under conditions thatresult in development of live offspring, thereby producing a non-mutantmouse.

[0017] A variety of methods can be used to introduce mouse pluripotentcells, such as non-inbred mouse ES cells, into mouse tetraploidblastocysts. In one embodiment, this is carried out by injecting thenon-inbred cells into tetraploid blastocysts, such as by microinjection,particularly piezo microinjection. Other methods can be used tointroduce non-inbred ES cells into the blastocysts. For example, themethod described by Amano et al., or a modification thereof, can be used(Amano, T. et al., Theriogenology 53, 1449-1458 (2000)). Alternatively,any other method, such as a chemical method, which results inintroduction of non-inbred ES cells into tetraploid blastocysts can beused.

[0018] Non-inbred pluripotent cells, such as non-inbred ES cells, usedin the present method can contain at least one/one or more geneticalterations or mutations. Alternatively, as described above, non-inbredES cells used can be non-mutant (have not been altered, after they areobtained, to contain a genetic alteration or mutation); such cells areused to produce non-mutant progeny by the method of the presentinvention. The genetic alterations or mutations that can be present innon-inbred ES cells used include, but are not limited to, transgenes(cDNA, genes or portions thereof), mutations (targeted or random),conditional mutations, targeted insertions of foreign genes, YAC and BACsized transgenes, all or part of a chromosome, which may be from thesame species as the embryo or another species, such as from a human.They include physical knockout of all or a part of a gene, functionalknockout of a gene, introduction of a functional gene and introductionof DNA or a gene portion that changes the function/level of expressionof a gene present in the ES cell (e.g., a promoter, enhancer orrepressor). An important feature of the method of the present inventionis that multiple genetic alterations, which will typically beconsecutive genetic alterations but can also be simultaneous, can bemade in the non-inbred ES cells, thus circumventing the need forbreeding to combine multiple alterations in one animal, as is requiredif presently-available methods are used. Alterations can also be presentin the non-inbred ES cells as they are obtained from the zygote fromwhich they are derived. As used herein, the terms mutant non-inbredpluripotent cells, mutant non-inbred ES cells and similar termsencompass cells which comprise a mutation or mutations as obtained fromthe zygote which gave rise to the cells and cells which are mutated oraltered after they are obtained from the zygote. Alterations can all beof the same type (e.g., all introduction of exogenous DNA) or of morethan one type (e.g., introduction of exogenous DNA, gene knockout andconditional gene knockout). They can also be a combination of mutationspresent in the non-inbred ES cells as derived from a zygote andmutations made after they are derived from a zygote. The alterationsmade in genomic DNA of non-inbred ES cells can be chosen to produce aphenotype that is similar to (mimics) a condition that occurs in otherspecies (e.g., humans) and the resulting mutant mice can, thus, serve asa model for that condition.

[0019] A variety of methods, known to those of skill in the art, can beused to alter or mutate inbred pluripotent (e.g., ES) cells or celllines to be used in the method of producing ES mice of the presentinvention. For example, an appropriate vector or plasmid can be used tointroduce DNA into ES cells in order, for example, to integrate DNA intogenomic DNA, express foreign DNA in recipient cells, cause recombination(homologous or nonhomologous) between introduced DNA and endogenous DNAor knock out endogenous gene(s), such as by means of the Cre-lox method.Alternatively, alterations or mutations can be produced by chemicalmethods or radiation. Gene targeting can also be used to produce mutantnon-inbred pluripotent cells or cell lines, such as mutant non-inbred EScells or cell lines. For example, the methodology described by Rideoutand co-workers can be used. See, Rideout, W. M. et al., Nature Genetics,24:109 (2000) and the references cited therein.

[0020] Tetraploid blastocysts can be produced by known methods, such asthat described by James and co-workers. James, R. M. et al, Genet. Res.Camb., 60:185 (1992). See also Wang, Z-Q et al, Mech. Dev., 62: 137(1997) and the references cited therein.

[0021] The invention also relates to methods of identifying XO F1 EScells, such as mouse XO F1 ES cells, by screening a population of F1 EScells, such as a population of wildtype or mutant F1 ES cells, for F1 EScells that are XO F1 ES cells. By population of F1 ES cells is meant amixture of XO F1 ES cells and XY F1 ES cells. In a particularembodiment, screening is carried out to identify F1 ES cells in whichspontaneous loss of the Y chromosome has occurred, resulting in XO F1 EScells. The invention further provides methods for isolating XO F1 EScells from XY F1 ES cells, comprising (a) screening a population of F1ES cells (e.g., wildtype or mutant) for loss of the Y chromosome; and(b) isolating F1 ES cells lacking the Y chromosome, thereby isolating XOF1 ES cells. Screening can be carried out using a variety of methodsknown and readily available in the art, such as using a Y chromosomeprobe (e.g., against repetitive elements) in Southern blot analysis orPCR amplification. F1 ES cells can be generated using a variety ofmethods known and readily available in the art, such as, for example, bylimiting dilution subcloning or transfection with exogenous DNA followedby appropriate selection. By exogenous DNA is meant any DNA that is notendogenous to the cells to be transfected or that does not already occurin the cells to be transfected. An example of an exogenous DNA is a drugselection marker.

[0022] Also the subject of this invention are mutant non-human mammals(e.g., mutant ES mice) produced by the method described herein; methodsof producing non-human mammalian embryos; non-human embryos produced bythe method; and a method of identifying a drug to be administered totreat a condition that occurs in a mammal, such as a human. The methodof producing mutant non-human mammalian embryos comprises injectingnon-human F1 ES cells into non-human tetraploid blastocysts andmaintaining the resulting tetraploid blastocysts under conditions thatresult in formation of embryos, thereby producing a mutant non-humanmammalian embryo(s). In one embodiment, the non-human mammalian embryois a mutant mouse embryo.

[0023] Another embodiment of the present invention is a method ofproducing a non-human mammalian strain, such as a mouse strain,particularly a mutant mouse strain, that is derived from a given(single) ES cell clone, such as a mutant non-inbred ES cell clone,without outcrossing with a wildtype partner. Until now, it has not beenpossible to do so because each F1 ES cell line is of a given sex(generally male). However, fertile XO females have been produced from amale ES cell clone by in vitro selection for loss of the Y chromosome.As a result, a mutant mouse strain carrying all genetic alterations canbe derived by breeding XY males and XO females, both derived from thesame targeted ES cell clone and of identical genetic makeup withoutoutbreeding of the mutant male to a normal female. Production of XY andXO subclones from the same clone, such as a clone carrying one or moregenetic mutation or a multiply targeted clone, makes it possible togenerate males and females carrying the same genetic alterations; suchmales and females can then be used to produce genetically identicaloffspring without the need for outbreeding.

[0024] For example, insertion of a negative selection marker (e.g., theHerpes Tk gene) on the Y chromosome of F1 ES cells makes it possible toderive XY and XO subclones from an initial male ES cell clone. The XO EScells can be produced, for example, by inserting a Herpes Tk gene ontothe Y chromosome by homologous recombination using known methods. Forexample, a vector that contains sequences homologous to a Y-linked gene(such as the Sry gene, the Mov15 gene or any other Y-linked gene) andexpresses the Tk gene can be produced and introduced into ES cells. Thecells are maintained under conditions that result in homologousrecombination between vector sequences and Y chromosome sequences. TheTk gene is, as a result, introduced into the Y chromosome. The resultingcells can then be targeted or otherwise genetically altered. To generatean XO line from the clones, the cells are subjected to selection byculturing in the presence of gancyclovir, which results in killing ofall cells carrying the Y chromosome into which the Tk gene has beeninserted. XO cells can be used to produce XO females, using knownmethods. Similarly, XY cells can be used to produce males. The resultingmales and females can be bred to produce offspring carrying the samegenetic material (mutations) as the parents. Other negative selectionmarkers, such as diphtheria toxin, can be used and are introduced intothe Y chromosome in a manner similar to that described for introductionof the Tk gene. Alternatively, XO ES cells can be produced byintroducing DNA into XY ES cells, such as by homologous recombination tofunctionally or physically delete the Y chromosome. The desired XO cellscan be identified, for example, by use of a Y chromosome probe (e.g.,for repetitive elements) in Southern blot analysis. In addition, becausethe Y chromosome is frequently lost spontaneously upon in vitro cultureof ES cells, the mutant male F1 ES cells can be passaged and subclonesscreened for spontaneous loss of the Y chromosome. XO ES cells can beidentified, as described in Example 3, for example, through Southernblot analysis in which a Y chromosome probe against repetitive elementsis used. XO ES cells can also be identified using other methods knownand readily available in the art, such as, for example, by PCR. Fertilefemale mice have been produced from male F1 ES cells that have a malekaryotype and are positive for Y-specific sequences by PCR in earlypassages. The B6×Balb line, V30. 11 has produced 4/4 females who, asjudged, for example, by their coat color, should be totally derived frommale ES cells.

[0025] The method of the present invention is, thus, also a method ofproducing a mutant mouse by breeding a mutant male mouse and a mutantfemale mouse, wherein the male mouse (or an ancestor thereof) and thefemale mouse (or an ancestor thereof) were produced from the same F1male ES cells or cell line, such as from the same targeted male ES cellclone, and the female mouse is an XO female. That is, the method is oneof producing a mutant mouse strain by breeding a mutant male mouse and amutant female mouse carrying identical genetic alterations as a resultof having been derived from a single targeted male F1 ES cell clone. Themutant female mouse is XO and is produced (or is the progeny of anancestor which was produced) by selecting for loss of the Y chromosomefrom a single (individual) male ES cell clone. The present inventionalso encompasses female mice which are XO and were produced from an F1male ES cell or cell line, such as by knocking out of the Y chromosome.It also encompasses progeny produced by breeding a male and a femaleproduced as described herein and progeny thereof.

[0026] The mutant non-human mammals, such as mutant mice, can be used asa model for a condition for which a preventive or therapeutic drug issought. A method of identifying a drug to be administered to treat acondition in a mammal comprises producing, using the method of thepresent invention, a mutant mouse that is a model of the condition;administering to the mutant mouse a drug, referred to as a candidatedrug, to be assessed for its effectiveness in treating or preventing thecondition; and assessing the ability of the drug to treat or prevent thecondition. If the candidate drug reduces the extent to which thecondition is present or progresses or causes the condition to reverse(partially or totally), the candidate drug is a drug to be administeredto treat the condition.

[0027] The present invention is illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES

[0028] The following examples describe production of mice using inbredES cells from four different ES cell lines from three inbred backgrounds(129/Sv, C57BL/6 and BALB/c) and six different F1 lines (129/Sv×C57BL/6,C57BL×129/Sv, BALB/c×129/Sv, 129/Sv×M. castaneus, C57BL/6×BALB/c and129/Sv×FVB); assessment of pups produced using the two types of EScells; and comparison of results obtained. The results show that use ofF1 ES cells consistently results in production of viable mice, whethertargeted or untargeted cells are used. They also demonstrate thatgenetic background is a crucial parameter for postnatal survival of pupsderived from ES cells. Further, they demonstrate that the method of thepresent invention has been successfully used to produce mice thatcontain desired alterations without the need to produce and breedchimera en route to producing the desired non-chimeric pups.

[0029] Methods and Materials

[0030] The following methods and materials were used to produce mousepups.

[0031] Production of ES Cell Clones

[0032] Nuclear transfer of ES cell nuclei into enucleated metaphase IIoocytes was carried out as previously described (Wakayama, T. et al.,Nature, 394:369-374 (1998); Wakayama, T. & Yanagimachi, R., NatureGenet., 22:127-128 (1999); Ogura, A. et al., Biol. Reprod., 62:1579-1584(2000); Rideout, W. M. et al., Nature Genet., 24:109-110(2000);Wakayama, T. et al., Proc. Natl. Acad. Sci. USA, 96: 14984-14989(1999)). 1-3 hours after nuclear transfer oocytes were activated for 5hours with 10 mM Sr⁺⁺ in Ca⁺⁺ free media in the presence of 5 mg/ml ofCytochalasin B. Embryos were cultured in vitro to the blastocyst stageand transferred to recipient mothers.

[0033] Embryo Culture

[0034] All embryo culture was carried out in microdrops on standardbacterial petri-dishes (Falcon) under mineral oil (Squibb). Modified CZBmedia (Chatot, C. L. et al., Biol. Reprod., 42: 432-440 (1990)) was usedfor embryo culture unless otherwise noted. Hepes buffered CZB was usedfor room temperature operations while long term culture was carried outin bicarbonate buffered CZB at 37° C. with an atmosphere of 5% CO₂ inair.

[0035] Recipient Females and Cesarean Section

[0036] Ten injected blastocysts were transferred to each uterine horn of2.5 days post coitum pseudopregnant Swiss females that had mated withvesectomized males. Recipient mothers were sacrificed at E 19.5 and pupswere quickly removed from the uterus. After cleaning fluid from theirair passages, pups were placed under a warming light and respiration wasobserved. Surviving pups were fostered to lactating BALB/c albinomothers.

[0037] Culture of Embryonic Stem (ES) Cells

[0038] Derivation and culture of embryonic stem cells were carried outas previously described (Nagy, A. et al., Development, 110:815-821(1990)) with ES cell lines derived from both inbred and F1 blastocysts.ES cells were cultured in DMEM with 15% FCS containing 1000 U/mlLeukocyte Inhibiting Factor (LIF) on gamma-irradiated primary feederfibroblasts. For blastocyst injection ES cells were trypsinized,resuspended in DMEM and preplated on a standard 10 cm tissue culturedish for thirty minutes to remove feeder cells and debris.

[0039] Preparation of Two Cell Embryos for Electrofusion

[0040] B6D2F1 females were superovulated by IP injection of 7.5 IU PMS(Calbiochem) followed 46-50 hours later with 7.5 IU HCG (Calbiochem).After administration of HCG, females were mated with B6D2F1 males.).Fertilized zygotes were isolated 24 hours later. Zygotes were left inHepes buffered CZB with 0.1% bovine testicular hyaluronidase for severalminutes at room temperature to remove any remaining cumulus cells. Afterwashing, zygotes were transferred to a new culture dish containing dropsof bicarbonate buffered CZB and placed at 37° overnight to obtaintwo-cell embryos.

[0041] Preparation of Tetraploid Embryos by Electrofusion

[0042] 40 hours post HCG the blastomeres of two-cell embryos wereelectrofused to produce one-cell tetraploid embryos. Electrofusion wascarried out on in inverted microscope using the lid of a petri dish as amicro-manipulation chamber. Platinum wires were used as both electrodesand micromanipulators to align two cell embryos for fusion. A group of15 two-cell embryos was placed on the stage in a 200 ml drop of M2 media(Sigma). Embryos were aligned with the interface between their twoblastomeres perpendicular to the electrical field and a singleelectrical pulse of 100V with a duration of 100 ms was applied to eachindividually. Manipulation of a single group took less then fiveminutes. After electrofusion, embryos were returned to CZB media at 37°C. Embryos that had not undergone membrane fusion within 1 hour werediscarded.

[0043] Piezo Micromanipulator Injection of Tetraploid Blastocyts

[0044] For microinjection, 5-6 blastocysts were placed in a drop of DMEMwith 15% FCS under mineral oil. A flat tip microinjection-pipette withan internal diameter of 12-15 um was used for ES cell injection. 15 EScells were picked up in the end of the injection pipette. The blastocystto be injected was held in the vicinity of the ICM with a standardholding pipette. The injection pipette, containing the ES cells waspressed against the zona opposite the inner cell mass. A brief pulse ofthe Piezo (Primatech Pmm, Ibaraki, Japan) was applied and the injectionneedle was simultaneously pushed through the zona and trophectodermlayer into the blastocoel cavity. The ES cells were then expelled fromthe injection pipette and pushed against the inner cell mass of theblastocyst. After injection of the entire group, blastocysts werereturned to CZB media and placed at 37° C. until transfer to recipientfemales.

Example 1 Assessment of the Effects of Genetic Heterogeneity of DonorCells on Development of ES Cell-tetraploid Pups

[0045] The possible effect of genetic heterogeneity of the donor cellson the development of ES cell-tetraploid pups was tested by transferringinbred or F1 ES cells into tetraploid blastocysts and assessingsurvival. Injection of ES cells into the blastocoel cavity of tetraploidblastocysts was aided by the use of a piezo-driven micromanipulator andthe resulting composite embryos were transferred to recipient females.312 tetraploid blastocysts were injected with four different inbred EScell lines that gave rise to 20 pups (6%) that were alive and active atcesarean section. However, 17 of the 20 newborns died of respiratoryfailure within 30 minutes. Of the three remaining pups, two were unableto sustain respiration and died within the next few hours (Table 1).Only one inbred ES cell-tetraploid pup was able to sustain respirationand developed to adulthood. In contrast, of 344 tetraploid blastocystsinjected with 6 different F1 ES cell lines, 60 (18%) developed to birth,51 of which (85%) survived to adulthood (Table 2). Thus, geneticheterogeneity of the donor ES cells has a significant effect onlong-term survival of both nuclear clones and ES cell-tetraploid pups.

[0046] It has been previously shown that continued passage of ES cellsis detrimental to their developmental potency (Wang, Z. Q., et al.,Mech. Dev., 62:137-145 (1997); Nagy, A. et al., Proc. Natl. Acad. Sci.USA, 90:8424-8428 (1993)). In order to assess whether continuous invitro culture would impair the survival of F1 ES cell-tetraploid pups, a129Sv×C57BL/6 ES cell line (V6.5) was kept continuously in culture andinjected into tetraploid blastocysts after prolonged passage. Noimpairment of postnatal survival of the resulting ES cell-tetraploidpups was noted after either 15 or 25 passages. In addition, F1 EScell-tetraploid mice were produced from cells that had been subjected totwo consecutive rounds of drug selection. First, selection withpuromycin was used for isolating cells that carried a targeted insertionof a tet-transactivator gene in the Rosa26 locus. Second, hygromycinselection was used to isolate cells with a tet-inducible promoterdriving expression of a hygromycin-thymidine kinase cassette in a randomlocus. Injection of these double-selected cells into 20 tetraploidblastocysts resulted in one fall-term pup, which survived to adulthood(Table 2). The results described herein indicate that live, adult mice,entirely derived from ES cells can be generated from F1 ES cells evenafter long-term passage of the cells in culture or after consecutiverounds of drug selection.

Example 2 Histological Assessment of Lungs

[0047] ES cell-tetraploid pups derived from inbred ES cells appeared tosuffer from respiratory distress after delivery. Histological analysisof both inbred and F1 completely ES cell derived neonates was carriedout. Examination of the lungs from inbred ES cell-tetraploid pupsrevealed that the alveoli were not inflated, while the lungs of newbornsderived from F1 ES cells were fully inflated. In addition, interstitialbleeding was often seen in inbred ES cell derived mice. Theseobservations suggest that the failure to initiate breathing and/orsustain normal circulation likely contributed to postnatal death ofinbred ES cell-tetraploid pups.

[0048] Results described herein demonstrate that genetic heterozygosityis a crucial parameter influencing postnatal survival of pups derivedfrom ES cells by tetraploid embryo complementation. Pups derived frominbred ES cells die perinatally with a phenotype of respiratory failure.In contrast, the great majority (80 to 85%) of pups derived from F1 EScells survived to adulthood. The observed respiratory phenotype appearsto be due to the inbred nature of the ES cell genome.

[0049] The possibility of deriving mice directly from ES cells withoutthe production of a chimeric intermediate has great potential forfacilitating the generation of animals with multiple geneticalterations. In conventional approaches, targeted ES cells are injectedinto diploid blastocysts to generate chimeric founders. The derivationof transgenic mice carrying the desired mutant allele requiresout-crossing these chimeras with wild type mice. Thus, the generation ofcompound animals that combine multiple desired alleles or transgenes intheir genome entails time-consuming and expensive cycles of crossingmice derived from different chimeric founders. In contrast, the EScell-tetraploid technology in combination with F1 ES cells allowsassembling multiple genetic alterations in the same ES cell line byconsecutive gene targeting cycles in vitro prior to generating mutantanimals. The desired transgenic mice with numerous genetic alterationscan be derived in a single step by injecting the multiply targeted F1 EScells into tetraploid blastocysts. Finally, unlike nuclear cloningtechnology, which has proven both difficult to master and transfer fromlaboratory to laboratory, the ES cell-tetraploid technology is easilyadapted to any laboratory currently creating chimeric mice by ES cellblastocyst injection.

[0050] At present, the mechanisms that permit long-term survival ofclones and ES cell-tetraploid pups derived from F1 but not from inbredES cells are unclear. Though it is generally assumed that “hybrid vigor”is an important parameter in animal survival under various selectiveconditions, it is not apparent whether wide-ranging chromosomalheterozygosity or heterozygosity at only a few crucial modifier loci isrequired. Examining the potency of ES cells that have been derived frombackcrosses between F1 mice and their parental inbred strains mayclarify this question. TABLE 1 Survival of Inbred ES Cell-TetraploidPups Pups Pups Pups respirating surviving alive at after to adult- EScell 4N blasts term C-section hood line Genotype injected (% Inj) (%Alive) (% Alive) J1 129/Sv 120  9 (7.5) 0 0 V18.6 129/Sv  48  5 (10)  1(20) 0 V26.2 C57BL/6  72  3 (4)   1 (33) 0 V39.7 BALB/c  72  3 (4)   1(33) 1 (33) Total Inbred 312 20 (6)   3 (15) 1 (5) 

Example 3 Generation of Fertile Mice Carrying A Mutation of Interest.

[0051] A male targeted F1 ES cell line was made as described above. Thecell line was then screened by Southern blot for subclones that havelost the Y chromosome. The probe used in the Southern blot to identifytargeted (mutant) ES subclones which have lost the Y chromosome was a Ychromosome probe against repetitive elements. This probe was previouslydescribed by Lamar, E. E. and Palmer, E., Cell, 37:171-177 (1984).

[0052] Subclones that have lost the Y chromosome were taken and used tomake mice by tetraploid embryo complementation as described above. Thefemale ES mice produced were shown to be fertile, indicating that theycan transmit the mutation of interest.

[0053] Male ES mice were produced from parent cell lines as describedabove. The male ES mice produced were shown to be fertile.

[0054] The loss of the Y chromosome, as shown by Southern blot analysis,is a general phenomenon in several ES lines, as demonstrated by theresults shown in Table 3. Loss of the Y chromosome occurs at a frequencythat can easily and routinely be screened for, as shown in Table 4.

[0055] Both male (generated from the Flpreporter targeted line) andfemale (generated from subclone #315) mice carry the exact mutation atthe same locus in the genome and when intercrossed will directly lead tohomozygous mutant off-spring. Both male and female ES mice were shown tobe fertile.

[0056] While this invention has been particularly shown and describedwith reference to particular embodiments thereof, it will be understoodby those skilled in the art that various changes inform and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims. TABLE 2 Survival of F1 ESCell-Tetraploid Pups Pups Pups Pups respirating surviving alive at afterto adult- ES cell 4N blasts term C-section hood line Genotype injected(% Inj) (% Alive) (% Alive) V6.5 C57BL/6 72 18 (25) 17 (94) 16 (89) X129/Sv V6.5* C57BL/6 60 11 (18)  9 (81)  9 (81) X 129/Sv V6.5** C57BL/620  1 (15)  1 (100)  1 (100) X 129/Sv 129B6 129/Sv 48 2 (4)  1 (50)  1(50) x C57BL/6 F1.2-3 129/Sv 48 4 (8)  3 (75)  3 (75) X M. Cast. V8.1129/Sv 24  7 (30)  7 (100)  7 (100) x FVB V17.2 BALB/c 48 13 (27) 12(92) 11 (85) X 129/Sv V30.11 C57BL/6 24  4 (30)  4 (100)  3 (75) XBALB/c Total F1 344  60 (18) 54 (90) 51 (85)

[0057] TABLE 3 Frequency of Y Chromosome Loss in WT and Targeted ES CellLines # of # of subclones subclones lacking Y screened for repeats EScell line Genotype Passage # loss of Y (% of screened) V6.6 129svJaeP8-9 448 8(1.8) x c57B6 B6129 C57B6 P6 146 4(2.7) x 129svJae J1 129svJaeP7 289 5(1.7) V6.5LJGF1 129svJae 1X 210 3(1.4) preport x trans c57B6fection

[0058] TABLE 4 Production of Male and Female ES-Tetraploid Mice fromTargeted Cell Lines 4N Neonates alive Mice Sex of surviving Blasts atC-Section Surviving to mice ES cell line* Inj (% blasts) AdulthoodFemale Male FLP reporter 68 7(10.2) 3(4.4) 0 3 FLP reporter Subclone #6663 7(11.1) 6(9.5) 6 0 FLP reporter Subclone #315 46 5(10.8) 5(10.8) 5 0# (#66 and #315), which were identified as having lost the Y chromosome,generated female mice.

What is claimed is:
 1. A method of producing a non-human mammal,referred to as an ES non-human mammal, wherein pluripotent cells areintroduced into tetraploid blastocysts of the same mammalian speciesunder conditions that result in production of an embryo and theresulting embryo is transferred into a foster mother which is maintainedunder conditions that result in development of live offspring, whereinthe pluripotent cells are non-inbred pluripotent cells.
 2. The method ofclaim 1, wherein the non-human mammal is a mouse.
 3. The method of claim2, wherein the pluripotent cells are embryonic stem cells and areintroduced into tetraploid blastocysts by injection.
 4. The method ofclaim 3, wherein injection is piezo microinjection.
 5. A method ofproducing a non-human mammalian embryo comprising injecting non-humannon-inbred ES cells into non-human tetraploid blastocysts andmaintaining the resulting tetraploid blastocysts under conditions thatresult in formation of embryos, thereby producing a non-human mammalianembryo.
 6. The method of claim 5, wherein the non-human non-inbred EScells are mouse cells and the non-human mammalian embryo is a mouse. 7.The method of claim 6, wherein mutant mouse non-inbred ES cells areinjected into non-human tetraploid blastocysts by piezo microinjection.8. A non-human mammal produced by the method of claim
 1. 9. A mouseproduced by the method of claim
 2. 10. A mouse produced by the method ofclaim
 3. 11. A non-human mammalian embryo produced by the method ofclaim
 5. 12. A mouse embryo produced by the method of claim
 6. 13. Amouse embryo produced by the method of claim
 7. 14. A method ofproducing a mutant non-human mammal, wherein pluripotent cellscomprising at least one mutation in genomic DNA are introduced intotetraploid blastocysts of the same mammalian species under conditionsthat result in production of an embryo and the resulting embryo istransferred into a foster mother which is maintained under conditionsthat result in development of live offspring, thereby producing a mutantnon-human mammal, wherein the pluripotent cells are non-inbredpluripotent cells.
 15. The method of claim 14, wherein the non-humanmammal is a mouse.
 16. The method of claim 15, wherein the pluripotentcells are embryonic stem cells and are introduced into tetraploidblastocysts by injection.
 17. The method of claim 16, wherein injectionis piezo microinjection.
 18. A method of producing a mutant non-humanmammalian embryo comprising injecting mutant non-human non-inbred EScells into non-human tetraploid blastocysts and maintaining theresulting tetraploid blastocysts under conditions that result information of embryos, thereby producing a mutant non-human mammalianembryo.
 19. The method of claim 18, wherein the mutant non-humannon-inbred ES cells are mouse cells and the mutant non-human mammal is amouse.
 20. The method of claim 19, wherein mutant mouse non-inbred EScells are injected into non-human tetraploid blastocysts by piezomicroinjection.
 21. A mutant non-human mammal produced by the method ofclaim
 14. 22. A mutant mouse produced by the method of claim
 15. 23. Amutant mouse produced by the method of claim
 16. 24. A mutant mouseembryo produced by the method of claim
 17. 25. A mutant mouse embryoproduced by the method of claim
 19. 26. A mutant mouse embryo producedby the method of claim
 20. 27. A method of producing a mutant mouse,comprising: (a) introducing mouse non-inbred ES cells comprising atleast one mutation in genomic DNA into mouse tetraploid blastocysts,thereby producing mouse blastocysts containing mouse non-inbred EScells; (b) maintaining the product of (a) under conditions that resultin production of embryos; (c) introducing an embryo into apseudopregnant female: and (d) maintaining the female into which theembryo is introduced under conditions that result in development of liveoffspring, thereby producing a mutant mouse.
 28. The method of claim 27,wherein in (a) introducing is carried out by injection.
 29. The methodof claim 28, wherein microinjection is piezo microinjection.
 30. Themethod of claim 29, wherein the at least one mutation in genomic DNA isa gene knockout or exogenous DNA incorporated into the genomic DNA. 31.A mouse embryo produced from a mouse tetraploid blastocyst havingincorporated therein mutant mouse non-inbred ES cells.
 32. The mouseembryo of claim 31, wherein the non-inbred ES cells are selected fromthe group consisting of: V6.5 cells; 129B6 cells; F1.2-3 cells; V8.1cells; V17.2 cells and V30.11 cells.
 33. The mouse embryo of claim 31,wherein the mutant mouse non-inbred ES cells comprise at least onemutation selected from the group consisting of: transgenes which arecDNA, genes or portions thereof; targeted mutations, random mutations,conditional mutations, targeted insertions of foreign genes, YAC sizedtransgenes, BAC sized transgenes, and all or part of a chromosome. 34.The mouse embryo of claim 33, wherein the at least one alteration ingenomic DNA is a gene knockout or exogenous DNA incorporated into thegenomic DNA.
 35. A method of producing a mouse, comprising: (a)introducing mouse non-inbred ES cells into mouse tetraploid blastocysts,thereby producing mouse blastocysts containing mouse non-inbred EScells; (b) maintaining the product of (a) under conditions that resultin production of embryos; (c) introducing an embryo into apseudopregnant female; and (d) maintaining the female into which theembryo is introduced under conditions that result in development of liveoffspring, thereby producing a mouse.
 36. The method of claim 35,wherein in (a) introducing is carried out by injection.
 37. The methodof claim 36, wherein microinjection is piezo microinjection.
 38. A mouseembryo produced from a mouse tetraploid blastocyst having incorporatedtherein mouse non-inbred ES cells.
 39. The mouse embryo of claim 38,wherein the non-inbred ES cells are selected from the group consistingof: V6.5 cells; 129B6 cells; F1.2-3 cells; V8.1 cells; V17.2 cells andV30.11 cells.
 40. A method of identifying a drug to be administered totreat a condition in a mammal in which the condition occurs, comprising:(a) producing, using the method of claim 14, a mutant mouse that is amodel of the condition; (b) administering to the mutant mouse a drug tobe assessed for its effectiveness in treating or preventing thecondition; and (c) assessing the ability of the drug to treat or preventthe condition, wherein if the drug reduces the extent to which thecondition is present or progresses, the drug is a drug to beadministered to treat the condition.
 41. A method of producing a mutantnon-human mammal, wherein pluripotent cells comprising at least onemutation in genomic DNA are introduced into tetraploid blastocysts ofthe same mammalian species under conditions that result in production ofan embryo and the resulting embryo is transferred into a foster motherwhich is maintained under conditions that result in development of liveoffspring, wherein the pluripotent cells are non-inbred pluripotentcells.
 42. A method of producing a mutant mouse that is derived from asingle non-inbred ES cell clone, comprising breeding a mutant male mouseand a mutant female mouse, wherein (a) the male mouse or an ancestorthereof and (b) the female mouse or an ancestor thereof were producedfrom the same non-inbred male ES cell and the female mouse is an XOfemale.
 43. The method of claim 42, wherein the non-inbred cell clone isa non-inbred F1 cell clone.
 44. A method of producing XO F1 ES cells,comprising: (a) introducing into male F1 ES cells a negative selectionmarker, under conditions appropriate for insertion of the negativeselection marker in the Y chromosome of male F1 ES cells, therebyproducing a mixture of male F1 ES cells comprising male F1 ES cells inwhich the negative selection marker is inserted in the Y chromosome andother male F1 ES cells, some of which do not contain a Y chromosome; and(b) subjecting the resulting mixture to conditions that result in thedeath of male F1 ES cells in which the Y chromosome has the negativeselection marker inserted therein and do not result in the death of maleF1 ES cells that lack a Y chromosome and are XO F1 ES cells, therebyproducing XO F1 ES cells.
 45. An XO female mouse produced by introducingXO F1 ES cells into tetraploid mouse blastocysts under conditions thatresult in production of an embryo and transferring the resulting embryointo a foster mother which is maintained under conditions that result indevelopment of live offspring, wherein the live offspring are XO femalemice.
 46. A method of producing a mutant mouse strain, comprisingbreeding a mutant male mouse and a mutant female mouse, wherein (a) themutant male mouse or an ancestor thereof and (b) the mutant female mouseor an ancestor thereof were derived from the same non-inbred male mouseES cell clone and the mutant female mouse is an XO female.
 47. Themethod of claim 46, wherein the non-inbred male ES cell is an F1 malemouse ES cell.
 48. The method of claim 47, wherein the mutant XO femalemouse or an ancestor thereof was derived from an male mouse F1 ES cellby knocking out the Y chromosome of the F1 ES cell, thereby producing anXO F1 ES cell; introducing the XO F1 ES cell into a tetraploid mouseblastocyst under conditions that result in production of an embryo andtransferring the resulting embryo into a foster mother which ismaintained under conditions that result in development of liveoffspring, thereby producing an XO female offspring.
 49. A method ofidentifying XO F1 ES cells, comprising screening a population of F1 EScells for F1 ES cells that are XO F1 ES cells, wherein said populationis a mixture of XO F1 ES cells and XY F1 ES cells.
 50. The method ofclaim 49, wherein screening is carried out to identify F1 ES cells inwhich spontaneous loss of the Y chromosome has occurred, therebyproducing XO F1 ES cells.
 51. The method of claim 50, wherein saidpopulation is a population of wildtype F1 ES cells or a population ofmutant F1 ES cells.
 52. The method of claim 51 wherein the F1 ES cellsare mouse cells.
 53. The method of claim 51, wherein screening iscarried out using a Y chromosome probe against repetitive elements orPCR amplification.
 54. A method of isolating XO F1 ES cells from XY F1ES cells, comprising (a) screening a population of F1 ES cells for lossof the Y chromosome, wherein said population is a mixture of XO F1 EScells and XY F1 ES cells; and (b) isolating F1 ES cells lacking the Ychromosome, thereby isolating XO F1 ES cells.
 55. The method of claim54, wherein said population is a population of wildtype F1 ES cells or apopulation of mutant F1 ES cells.
 56. The method of claim 55, whereinthe F1 ES cells are mouse cells.
 57. The method of claim 55, whereinscreening is carried out using a Y chromosome probe against repetitiveelements or PCR amplification.
 58. The method of claim 55, wherein theF1 ES cells is generated by introducing into male F1 ES cells a negativeselection marker under conditions appropriate for insertion of thenegative selection marker in the Y chromosome of male F 1 ES cells,thereby producing a mixture of F1 ES cells comprising male F1 ES cellsin which the negative selection marker is inserted in the Y chromosomeand other male F1 ES cells, some of which do not contain a Y chromosome;and subjecting the resulting F1 ES cells to conditions that result inthe death of male F1 ES cells in which the Y chromosome has the negativeselection marker inserted therein and do not result in the death of maleF1 ES cells that lack a Y chromosome.
 59. A method of producing an XOfemale non-human mammal, comprising introducing XO F1 ES cellsidentified using the method of claim 49 into tetraploid blastocysts ofthe same mammalian species under conditions that result in production ofan embryo and transferring the resulting embryo into a foster motherwhich is maintained under conditions that result in development of liveoffspring, thereby producing an XO female non-human mammal.
 60. Themethod of claim 59, wherein said XO F1 ES cells are wildtype XO F1 EScells or mutant XO F1 ES cells.
 61. The method of claim 44, wherein saidXO F1 ES cells are mouse cells and the non-human mammal is a mouse. 62.A non-human mammal produced by the method of claim
 59. 63. A non-humanmammal produced by the method of claim
 60. 64. A mouse produced by themethod of claim
 61. 65. A method of producing an XO female mouse,comprising introducing mouse XO F1 cells identified using the method ofclaim 49 into tetraploid mouse blastocysts under conditions that resultin production of an embryo and transferring the resulting embryo into afoster mother which is maintained under conditions that result indevelopment of live offspring, thereby producing an XO female mouse. 66.The method of claim 65, wherein the mouse XO F1 ES cells are wildtypemouse XO F1 ES cells or mouse mutant XO F1 ES cells.
 67. A mouseproduced by the method of claim
 65. 68. A mouse produced by the methodof claim
 66. 69. An XO female mouse produced by (a) introducing mouse XOF1 ES cells into tetraploid mouse blastocysts under conditions thatresult in production of an embryo, said mouse XO F1 ES cells identifiedby screening a population of mouse F1 ES cells for F1 ES cells that areXO F1 ES cells, wherein said population is a mixture of mouse XO F1 EScells and mouse XY F1 ES cells; and (b) transfecting the resultingembryo into a foster mother which is maintained under conditions thatresult in development of live offspring, wherein the live offspring areXO female mice.
 70. The XO female mouse of claim 69, wherein screeningis carried out to identify F1 ES cells in which spontaneous loss of theY chromosome has occurred, thereby producing XO F1 ES cells.
 71. The XOfemale mouse of claim 70, wherein said population of mouse F1 ES cellsis a population of mouse wildtype F1 ES cells or a population of mousemutant F1 ES cells.
 72. The method of claim 70, wherein screening iscarried out using a Y chromosome probe against repetitive elements orPCR amplification.